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The H2-receptor antagonist ranitidine interferes with clopidogrel-mediated P2Y12 inhibition in platelets
Pharmacological Research 62 (2010) 352–356
Contents lists available at ScienceDirect
Pharmacological Research journal homepage: www.elsevier.com/locate/yphrs
The H2 -receptor antagonist ranitidine interferes with clopidogrel-mediated P2Y12 inhibition in platelets Andreas Schäfer ∗ , Ulrike Flierl, Stephanie Pförtsch, Nora Seydelmann, Jan Micka, Johann Bauersachs Medizinische Klinik und Poliklinik I, Universitätsklinikum Würzburg, Julius-Maximilians-Universität Würzburg, Oberdürrbacher Str. 6, 97080 Würzburg, Germany
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Article history: Received 27 January 2010 Received in revised form 26 May 2010 Accepted 26 May 2010 Keywords: ADP receptors Platelet activation Ranitidine PPI P2Y12
a b s t r a c t Background: Use of proton-pump inhibitors (PPIs) is common in patients on dual antiplatelet therapy (DAT). Recent warnings about potential interactions of PPIs with clopidogrel metabolism leading to impaired DAT efficacy has prompted the recommendation of substituting PPIs with H2 -receptor antagonists such as ranitidine. We investigated whether ranitidine interacts with P2Y12 inhibition on the platelet level. Methods: Blood was collected from 15 patients with stable coronary artery disease, who had undergone elective coronary intervention. Clopidogrel responsiveness was assessed 24 h after the administration of a 600 mg loading dose using the P2Y12 specific platelet-reactivity-index (PRI) and light-transmittance aggregometry in the presence and absence of a pharmacologically relevant concentration of the H2 receptor antagonist ranitidine (400 ng/ml). Results: Adding ranitidine enhanced P2Y12 -mediated platelet reactivity to ADP assessed by the PRI (mean PRI ± SEM: before ranitidine 28 ± 5%; after ranitidine 37 ± 5%, p = 0.0025). Similarly, prostaglandin E1 (PGE1 )-mediated inhibition of ADP-induced aggregation was abrogated in the presence of ranitidine (Aggmax ± SEM: before PGE1 41 ± 2%; after PGE1 29 ± 2%, p < 0.01 vs. before PGE1 ; after PGE1 + ranitidine 42 ± 2%, p < 0.01 vs. after PGE1 ). Conclusions: Exposition of platelets with ranitidine significantly enhanced their responsiveness to ADP and contributed to impaired P2Y12 inhibition suggesting that ranitidine interacts with clopidogrel efficacy through adenylyl cyclase inhibition on the platelet level. © 2010 Elsevier Ltd. All rights reserved.
1. Introduction Dual antiplatelet therapy (DAT) with the P2Y12 inhibitor clopidogrel in addition to acetylsalicylic acid (ASA) is the standardof-care in patients following percutaneous coronary interventions (PCI). Following elective PCI, it is recommend that clopidogrel 75 mg daily should be given for at least 4 weeks after stent implantation with a 300 mg loading dose of clopidogrel at least 6 h prior to PCI [1,2]. To prevent gastrointestinal bleeding in patients on DAT [3], use of proton-pump inhibitors (PPIs) is common in those patients reaching treatment rates of 60–70% in real life populations [4,5].
Abbreviations: AC, adenylyl cyclase; ASA, acetylsalicylic acid; CAD, coronary artery disease; DAT, dual antiplatelet therapy; EMEA, European Medicines Agency; FDA, Food and Drug Administration; P2Y12 , ADP receptor P2Y12 ; PCI, percutaneous coronary interventions; PGE1 , prostaglandin E1; PPI, proton-pump inhibitor; PPP, platelet-poor plasma; PRI, platelet-reactivity-index; PRP, platelet-rich plasma; VASP, vasodilator-stimulated phosphoprotein. ∗ Corresponding author. Tel.: +49 931 201 1; fax: +49 931 201 639110. E-mail address: [email protected] (A. Schäfer). 1043-6618/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.phrs.2010.05.006
Clopidogrel inhibits multiple pro-aggregatory actions of the platelet agonist ADP by selectively and irreversibly blocking the P2Y12 platelet ADP receptor. However, following absorption, 85% of the inactive prodrug clopidogrel are already hydrolyzed by circulating blood esterases to an inactive metabolite before the remaining 15% undergo hepatic metabolization by cytochrome P450 isoenzymes such as CYP2C19 to generate the active metabolite [6]. This active metabolite forms disulfide bridges with the extracellular part of the P2Y12 receptor and inactivates the receptor in this way irreversibly. Large clinical trials demonstrated the superiority of adding clopidogrel to aspirin for preventing ischemic events in unstable angina [7]. Nevertheless, some patients experience thromboembolic events despite daily DAT, and so-called clopidogrel “non-responsiveness” has been observed [8–10]. When impaired responsiveness is defined based on the risk for potential stent thrombosis, a high proportion of patients in a real-world setting are suboptimal responders to clopidogrel in daily practice [5]. Genetic predisposition, e.g. reduced function of CYP2C19, has been highlighted as a contributor to impaired clopidogrel pharmacokinetics and–dynamics translating into increased cardiovascular risk [11–13]. Certain PPIs such as omeprazole but not pantoprazole
A. Schäfer et al. / Pharmacological Research 62 (2010) 352–356
strongly inhibit CYP2C19 and impair clopidogrel responsiveness [14–16]. Based on these data, American (FDA) and European (EMEA) regulatory authorities recently issued warnings about potential interactions of PPIs with clopidogrel metabolism leading to impaired DAT efficacy and recommended substituting PPIs with H2 -receptor antagonists. However, there are no data ensuring us to use H2 -receptor blockers in the context of clopidogrel-based DAT. Therefore, we assessed whether P2Y12 -inhibition would be affected by ranitidine. 2. Methods 2.1. Study population For the in vitro experiments on ranitidine interaction with clopidogrel, blood samples were collected from 15 patients with stable CAD 24 h after receiving a 600 mg clopidogrel loading dose for elective PCI (Table 1). For in vitro experiments in the absence of clopidogrel pretreatment, blood samples were also collected from 7 healthy donors. The study was performed in accordance with the Declaration of Helsinki. 2.2. Blood sample collection Blood samples were processed within 1 h after collection from an antecubital vein using a 21-G needle. The first 5 mL of blood were discarded to avoid spontaneous platelet activation. Platelet-rich plasma (PRP) was prepared from citrated blood by centrifugation at 180 × g for 10 min. The platelet count in PRP was adjusted to 250,000/L by dilution with platelet-poor plasma (PPP), which had been obtained by centrifugation of the blood fraction at 2000 × g for 10 min. PRP was kept at 37 ◦ C before use. 2.3. Platelet aggregation Platelet aggregation was performed with light-transmittance aggregometry using a commercial 8-channel platelet aggregation profiler (PAP-8, BioData, Horsham, PA). Light transmission was adjusted to 0% with PRP and to 100% using PPP for each measurement. Curves were recorded for 6 min and aggregation was measured at maximal aggregation (Aggmax ). For co-incubation with ranitidine, PRP was incubated with ranitidine (400 ng/ml) or buffer for 10 min before prostaglandin E1 (PGE1 ) incubation and subsequent stimulation with ADP. Table 1 Patient characteristics. Patients N Sex (m/f, m[%]) Age (years) LDL-C (mg/dl) HDL-C (mg/dl) HbA1c (%) MDRD (ml/min/1.73 m2 ) BMI (kg/m2 ) Hypertension (%) Hyperlipoproteinaemia (%) Diabetes mellitus (%) Smoking (%) Family history of CAD (%) Adiposity (%) CVRF (n)
15 10/5 (67) 62 ± 3 99.7 ± 7.0 45.9 ± 3.5 6.2 ± 0.2 90.8 ± 4.2 27.8 ± 0.9 73 67 20 67 40 53 3 (IQR 2–4)
LDL-C = low-density lipoprotein cholesterol; HDL-C = high-density lipoprotein cholesterol; MDRD = glomerular filtration rate estimated using the MDRD Equation (Modification of Diet in Renal Disease Study Group); BMI = body-mass index.
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2.4. Platelet-reactivity-index (PRI) The P2Y12 PRI was determined through assessment of the phosphorylation status of the vasodilator-stimulated phosphoprotein (VASP) using flow cytometry (FACSCalibur, Becton Dickinson, Heidelberg, Germany). VASP phosphorylation (VASP-P) was quantified with labelled monoclonal antibodies using a commercially available kit (PLT VASP/P2Y12 Test kit, BioCytex, Marseille, France, #7014), for which platelets were stimulated with PGE1 in the presence and absence of ADP followed by fixation with formaldehyde. Following permeabilization with Triton X-100, VASP-P at serine 239 was determined using a monoclonal antibody (16C2) and a FITC-labelled secondary antibody, while platelets were counterstained with a PE-labelled anti-CD61 antibody. For dilutions, all antibodies of the test kit were used as by the manufacturer’s instructions. The PRI was calculated after measurement of VASP-P levels following stimulation with PGE1 (0.5 mol/L) by mean fluorescence intensity (MFI PGE1 ) as well as stimulation with PGE1 in the presence of ADP (20 mol/L, MFI PGE1 + ADP). The P2Y12 PRI is defined as: ([MFI PGE1 ] − [MFI PGE1 + ADP])/[MFI PGE1 ] × 100%, whereby background fluorescence is subtracted from each measurement. The lower the P2Y12 PRI is the better clopidogrel inhibits P2Y12 activity. For assessment of ranitidine interaction, the complete experiment was run in parallel in the presence of a pharmacologically relevant ranitidine concentration (400 ng/ml). 2.5. Substances Unless stated otherwise, chemicals were obtained from Sigma (Deisenhofen, Germany) in the highest purity available. 2.6. Statistics Individual measurements are shown. Statistical analysis was performed by one-way ANOVA followed by Tukey’s multiple comparisons test or a paired Student’s t-test where applicable; P < 0.05 was considered statistically significant. Data were analyzed using GraphPad Prism 4.0 (GraphPad Software, Inc., La Jolla, CA). 3. Results 3.1. Influence of H2 -receptor antagonism in platelets from healthy donors In PRP from healthy donors, ranitidine attenuated the aggregation-inhibiting effect of prostaglandin E1 (PGE1 ). While ADP-induced aggregation (91.6 ± 4.1%, trace 1 in Fig. 1A) was not significantly affected by ranitidine (92.5 ± 4.2%, trace 2 in Fig. 1A), PGE1 alone reduced ADP-induced aggregation (21.1 ± 1.8%, p < 0.001, trace 3 in Fig. 1A). Pre-incubation of PRP with the relevant concentration of ranitidine (400 ng/ml) led to an aggregation equivalent to that observed in the absence of both PGE1 and ranitidine (81.4 ± 9.0%, p < 0.001, trace 4 in Fig. 1A). Thus, blocking the adenylyl cyclase-stimulating H2 -receptor with ranitidine counteracts the platelet-inhibiting properties of PGE1 (Fig. 1A and B). 3.2. Influence of H2 -receptor antagonism in platelets from clopidogrel-treated patients Similar effects were observed in PRP obtained from clopidogreltreated CAD patients, in which pre-incubation with ranitidine abrogated the aggregation-inhibiting effect of PGE1 (Fig. 1C). While ADP-induced aggregation (41.2 ± 2.3%) was significantly reduced by PGE1 alone (29.5 ± 2.0%, p < 0.001), pre-incubation of PRP with the relevant concentration of ranitidine (400 ng/ml) led to an aggregation equivalent to that observed in the absence of both PGE1
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Fig. 1. Modulation of prostaglandin E1 (PGE1 )-mediated inhibition of ADP-induced maximal aggregation (Aggmax ) by ranitidine (Rani, 400 ng/ml) in PRP from clopidogrelnaïve healthy volunteers (A). Representative aggregation response curves from a healthy donor were recorded in the presence of ADP only (A, trace 1), ADP + RANI (A, trace 2), ADP + PGE1 (A, trace 3), and ADP + RANI + PGE1 (A, trace 4). The quantitative analysis is shown for healthy donors (n = 7, B) and for similar experiments in blood from clopidogrel-treated patients with coronary artery disease 24 h after receiving a 600 mg clopidogrel loading dose (n = 15, C).
Fig. 2. Adenosine-5 -diphosphate (ADP) activates platelets through two G protein-coupled P2 receptors, P2Y1 and P2Y12 . The G␣q -coupled P2Y1 receptor initiates ADPinduced aggregation via phospholipase C (PLC) and mobilization of calcium (Ca2+ ), while the G␣i -coupled P2Y12 receptor mediates amplification and completion of the aggregation response. In addition, P2Y12 is linked to the inhibition of cyclic adenosine monophosphate (cAMP) production by adenylyl cyclase (AC), which can be stimulated by prostacyclin (PGI2 ), prostaglandin E1 (PGE1 ) or histamine through specific Gs -coupled receptors, the isoprostane receptor (IP), PGE1 -receptor (EP2), and the histamine-2 receptor (H2 R), respectively. cAMP-activated proteinkinase A (PKA) phosphorylates the vasodilator-stimulated phosphoprotein (VASP), which inhibits platelet aggregation and secretion. The extent of ADP-induced attenuation of PGE1 -mediated VASP phosphorylation is used in the platelet-reactivity-index to determine P2Y12 -specific ADPactivity. Thienopyridines and new ADP-antagonists inhibit multiple pro-aggregatory actions of ADP, mostly by preventing the P2Y12 -mediated secondary ADP-response contributing to amplification of platelet activation. Blockade of H2 R suppresses stimulation of AC and leads to a shift of the AC pathway towards activation.
and ranitidine (41.5 ± 2.4%, p < 0.001). Similarly, addition of ranitidine enhanced ADP-induced platelet aggregation in blood from clopidogrel-treated patients (45.8 ± 2.3%, p < 0.01 vs. ADP in the absence of ranitidine). This indicates, that modulating H2 -receptor signaling, which is a Gs -protein-coupled receptor activating adenylyl cyclase and, thereby, counteracting P2Y12 (Fig. 2), can interact with clopidogrel efficacy on a platelet level. When adding pharmacologically relevant levels of ranitidine to blood samples from clopidogrel-loaded CAD patients, the PRI displaying specifically P2Y12 -dependent platelet reactivity was sig-
nificantly increased from 34.1 ± 5.5% to 44.1 ± 5.1% (p = 0.0005, Fig. 3). 4. Discussion In the present study we found that in platelets from patients on DAT, the H2 -receptor blocker ranitidine enhanced ADP-induced and P2Y12 -mediated platelet signaling. High variability in clopidogrel responsiveness has been widely described [17]. Using P2Y12 -specific assays to assess clopidogrel responsiveness such as the PRI, suboptimal responses in up to 60%
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Fig. 3. ADP-induced platelet signaling in clopidogrel-treated stable CAD patients (n = 15) 24 h after receiving a 600 mg loading dose in the absence and presence of a pharmacologically relevant concentration of ranitidine (400 ng/ml) was determined by ADP-mediated inhibition of PGE1 -induced VASP phosphorylation assessed by the PRI. The typical flow-cytometric histogram shows the maximum PGE1 -response (filled grey), the background signal (black curve), and the ADP-induced reactivity in the absence (blue curve) and presence (red curve) of ranitidine. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
of patients have been described [5]. In particular, a PRI >50% is associated with increased stent thrombosis and major adverse cardiovascular events [18–21]. Especially patients with high platelet reactivity despite receiving a 600 mg loading dose of clopidogrel had worse outcome after elective PCI [22]. Increasing clopidogrel dosage in patients undergoing PCI to achieve a PRI below 50% reduces subsequent cardiovascular events [23]. Since genetic loss-of-function of CYP2C19 is related to impaired pharmacokinetic and pharmacodynamic response to clopidogrel as well as impaired outcome [11–13], medications exerting profound CYP2C19 inhibition might reasonably be considered as a unfavorable option in patients on DAT. The PPI omeprazole is a profound inhibitor of CYP2C19 [24], and was identified in a prospective functional study to significantly interact with clopidogrel and reduce its platelet inhibitory effect [14]. In this double-blind placebo-controlled trial, patients undergoing PCI were randomized to receive either omeprazole or placebo for 7 days in addition to DAT. Omeprazole significantly decreased the ability of clopidogrel to inhibit platelet reactivity assessed by the P2Y12 -specific PRI [14]. Several retrospective observational studies showed conflicting results with regard to PPIs and clopidogrel. In a populationbased case–control study, current PPI use was associated with an increased risk of reinfarction, however, pantoprazole, which does not inhibit CYP2C19, had no association with readmission for myocardial infarction [25]. A retrospective cohort study of >8000 patients with ACS taking clopidogrel after discharge showed that concomitant use of clopidogrel and PPIs was associated with an increased risk of adverse outcomes than use of clopidogrel without PPIs. However, almost all patients using a single PPI throughout the observation period had been on omeprazole [4] Furthermore, one un-published analysis indicated a potential risk in all patients treated with any PPI [26], whereas more recent insurance data from other sources showed no such clear effect [27]. In a previous study, we observed no difference of clopidogrel responsiveness in stable CAD patients on clopidogrel maintenance therapy in the presence of pantoprazole [5]. Data from three retrospective evaluations (not even registries) prompted the FDA and EMEA to state their warnings with regard to combining PPIs with clopidogrel [4,25,26]. The retrospective analysis of an insurance database demonstrated the strongest influence
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of PPIs on outcomes, however, there was an enormous mismatch of patients taking PPIs vs. those not taking PPIs [26]. The PPI cohorts incorporated all known relevant clinical risk factors for impaired clopidogrel responsiveness [28]. Of interest, recent secondary analyses from large randomized controlled trials comparing clopidogrel vs. active competitors in more homogenous patient collectives did not find differences in clinical outcomes when comparing PPItreated vs. not PPI-treated patients [29]. From an underpowered group in one of the insurance databases, it was actually recommended to substitute PPIs with H2 -receptor blockers for gastric ulcer prevention [26]. Again FDA and EMEA both stated that there were no data indicating an interaction. However, literature research revealed a paper describing that concomitant ranitidine treatment in acetylsalicylic acid-treated patients enhances ADP-induced aggregation, which is intended to be blocked by clopidogrel, and reduces circulating salicylate levels [30], thereby potentially interacting with both components of DAT. Indeed, our in vitro data on P2Y12 -specific signaling demonstrates that ranitidine interacts with P2Y12 -mediated signaling on the platelet level (Fig. 1), thereby contributing to impaired functional clopidogrel-responsiveness (Fig. 3). One P2Y12 -specific signaling of ADP is the inhibition of adenynyl cyclase through a G␣i . Adenynyl cyclase is constitutively stimulated by endogenous platelet inhibitors such as prostacyclin and prostaglandin E1 through Gs -protein-coupled receptors. Histamine acts on platelets through a similar Gs -protein-coupled receptor, the H2 receptor. By blocking this receptor, the balance is shifted towards less stimulation of adenylyl cyclase and, therefore, an ADP-like effect (Fig. 2). Due to the mechanism of P2Y12 -mediated and H2 -mediated signaling, a clopidogrel-counteracting effect of ranitidine is not surprising. This mechanism is absolutely distinct from potential effects of ranitidine by heightening gastric pH leading to reduced release of clopidogrel from the oral formulation or potential interactions with hepatic clopidogrel metabolism, which were beyond the scope of this investigation. Furthermore, a recent pharmacological assessment described that ranitidine affected the pharmacokinetics of clopidogrel maintenance dosing as well as prasugrel loading in patients treated with ranitidine [31]. It can be assumed, that other H2 -receptor blockers will have the same effect on the platelet level, however, cimetidine should not be considered as an appropriate substitution for PPIs, as its CYP2C19 inhibiting properties were even mentioned in the EMEA warnings. In conclusion, we demonstrate that ranitidine, which has been suggested as an alternative to PPIs in patients on DAT, does interfere with clopidogrel on the platelet level and should, therefore, not be recommended in the absence of prospective data as a comedication to clopidogrel.
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Contents lists available at ScienceDirect
Pharmacological Research journal homepage: www.elsevier.com/locate/yphrs
The H2 -receptor antagonist ranitidine interferes with clopidogrel-mediated P2Y12 inhibition in platelets Andreas Schäfer ∗ , Ulrike Flierl, Stephanie Pförtsch, Nora Seydelmann, Jan Micka, Johann Bauersachs Medizinische Klinik und Poliklinik I, Universitätsklinikum Würzburg, Julius-Maximilians-Universität Würzburg, Oberdürrbacher Str. 6, 97080 Würzburg, Germany
a r t i c l e
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Article history: Received 27 January 2010 Received in revised form 26 May 2010 Accepted 26 May 2010 Keywords: ADP receptors Platelet activation Ranitidine PPI P2Y12
a b s t r a c t Background: Use of proton-pump inhibitors (PPIs) is common in patients on dual antiplatelet therapy (DAT). Recent warnings about potential interactions of PPIs with clopidogrel metabolism leading to impaired DAT efficacy has prompted the recommendation of substituting PPIs with H2 -receptor antagonists such as ranitidine. We investigated whether ranitidine interacts with P2Y12 inhibition on the platelet level. Methods: Blood was collected from 15 patients with stable coronary artery disease, who had undergone elective coronary intervention. Clopidogrel responsiveness was assessed 24 h after the administration of a 600 mg loading dose using the P2Y12 specific platelet-reactivity-index (PRI) and light-transmittance aggregometry in the presence and absence of a pharmacologically relevant concentration of the H2 receptor antagonist ranitidine (400 ng/ml). Results: Adding ranitidine enhanced P2Y12 -mediated platelet reactivity to ADP assessed by the PRI (mean PRI ± SEM: before ranitidine 28 ± 5%; after ranitidine 37 ± 5%, p = 0.0025). Similarly, prostaglandin E1 (PGE1 )-mediated inhibition of ADP-induced aggregation was abrogated in the presence of ranitidine (Aggmax ± SEM: before PGE1 41 ± 2%; after PGE1 29 ± 2%, p < 0.01 vs. before PGE1 ; after PGE1 + ranitidine 42 ± 2%, p < 0.01 vs. after PGE1 ). Conclusions: Exposition of platelets with ranitidine significantly enhanced their responsiveness to ADP and contributed to impaired P2Y12 inhibition suggesting that ranitidine interacts with clopidogrel efficacy through adenylyl cyclase inhibition on the platelet level. © 2010 Elsevier Ltd. All rights reserved.
1. Introduction Dual antiplatelet therapy (DAT) with the P2Y12 inhibitor clopidogrel in addition to acetylsalicylic acid (ASA) is the standardof-care in patients following percutaneous coronary interventions (PCI). Following elective PCI, it is recommend that clopidogrel 75 mg daily should be given for at least 4 weeks after stent implantation with a 300 mg loading dose of clopidogrel at least 6 h prior to PCI [1,2]. To prevent gastrointestinal bleeding in patients on DAT [3], use of proton-pump inhibitors (PPIs) is common in those patients reaching treatment rates of 60–70% in real life populations [4,5].
Abbreviations: AC, adenylyl cyclase; ASA, acetylsalicylic acid; CAD, coronary artery disease; DAT, dual antiplatelet therapy; EMEA, European Medicines Agency; FDA, Food and Drug Administration; P2Y12 , ADP receptor P2Y12 ; PCI, percutaneous coronary interventions; PGE1 , prostaglandin E1; PPI, proton-pump inhibitor; PPP, platelet-poor plasma; PRI, platelet-reactivity-index; PRP, platelet-rich plasma; VASP, vasodilator-stimulated phosphoprotein. ∗ Corresponding author. Tel.: +49 931 201 1; fax: +49 931 201 639110. E-mail address: [email protected] (A. Schäfer). 1043-6618/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.phrs.2010.05.006
Clopidogrel inhibits multiple pro-aggregatory actions of the platelet agonist ADP by selectively and irreversibly blocking the P2Y12 platelet ADP receptor. However, following absorption, 85% of the inactive prodrug clopidogrel are already hydrolyzed by circulating blood esterases to an inactive metabolite before the remaining 15% undergo hepatic metabolization by cytochrome P450 isoenzymes such as CYP2C19 to generate the active metabolite [6]. This active metabolite forms disulfide bridges with the extracellular part of the P2Y12 receptor and inactivates the receptor in this way irreversibly. Large clinical trials demonstrated the superiority of adding clopidogrel to aspirin for preventing ischemic events in unstable angina [7]. Nevertheless, some patients experience thromboembolic events despite daily DAT, and so-called clopidogrel “non-responsiveness” has been observed [8–10]. When impaired responsiveness is defined based on the risk for potential stent thrombosis, a high proportion of patients in a real-world setting are suboptimal responders to clopidogrel in daily practice [5]. Genetic predisposition, e.g. reduced function of CYP2C19, has been highlighted as a contributor to impaired clopidogrel pharmacokinetics and–dynamics translating into increased cardiovascular risk [11–13]. Certain PPIs such as omeprazole but not pantoprazole
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strongly inhibit CYP2C19 and impair clopidogrel responsiveness [14–16]. Based on these data, American (FDA) and European (EMEA) regulatory authorities recently issued warnings about potential interactions of PPIs with clopidogrel metabolism leading to impaired DAT efficacy and recommended substituting PPIs with H2 -receptor antagonists. However, there are no data ensuring us to use H2 -receptor blockers in the context of clopidogrel-based DAT. Therefore, we assessed whether P2Y12 -inhibition would be affected by ranitidine. 2. Methods 2.1. Study population For the in vitro experiments on ranitidine interaction with clopidogrel, blood samples were collected from 15 patients with stable CAD 24 h after receiving a 600 mg clopidogrel loading dose for elective PCI (Table 1). For in vitro experiments in the absence of clopidogrel pretreatment, blood samples were also collected from 7 healthy donors. The study was performed in accordance with the Declaration of Helsinki. 2.2. Blood sample collection Blood samples were processed within 1 h after collection from an antecubital vein using a 21-G needle. The first 5 mL of blood were discarded to avoid spontaneous platelet activation. Platelet-rich plasma (PRP) was prepared from citrated blood by centrifugation at 180 × g for 10 min. The platelet count in PRP was adjusted to 250,000/L by dilution with platelet-poor plasma (PPP), which had been obtained by centrifugation of the blood fraction at 2000 × g for 10 min. PRP was kept at 37 ◦ C before use. 2.3. Platelet aggregation Platelet aggregation was performed with light-transmittance aggregometry using a commercial 8-channel platelet aggregation profiler (PAP-8, BioData, Horsham, PA). Light transmission was adjusted to 0% with PRP and to 100% using PPP for each measurement. Curves were recorded for 6 min and aggregation was measured at maximal aggregation (Aggmax ). For co-incubation with ranitidine, PRP was incubated with ranitidine (400 ng/ml) or buffer for 10 min before prostaglandin E1 (PGE1 ) incubation and subsequent stimulation with ADP. Table 1 Patient characteristics. Patients N Sex (m/f, m[%]) Age (years) LDL-C (mg/dl) HDL-C (mg/dl) HbA1c (%) MDRD (ml/min/1.73 m2 ) BMI (kg/m2 ) Hypertension (%) Hyperlipoproteinaemia (%) Diabetes mellitus (%) Smoking (%) Family history of CAD (%) Adiposity (%) CVRF (n)
15 10/5 (67) 62 ± 3 99.7 ± 7.0 45.9 ± 3.5 6.2 ± 0.2 90.8 ± 4.2 27.8 ± 0.9 73 67 20 67 40 53 3 (IQR 2–4)
LDL-C = low-density lipoprotein cholesterol; HDL-C = high-density lipoprotein cholesterol; MDRD = glomerular filtration rate estimated using the MDRD Equation (Modification of Diet in Renal Disease Study Group); BMI = body-mass index.
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2.4. Platelet-reactivity-index (PRI) The P2Y12 PRI was determined through assessment of the phosphorylation status of the vasodilator-stimulated phosphoprotein (VASP) using flow cytometry (FACSCalibur, Becton Dickinson, Heidelberg, Germany). VASP phosphorylation (VASP-P) was quantified with labelled monoclonal antibodies using a commercially available kit (PLT VASP/P2Y12 Test kit, BioCytex, Marseille, France, #7014), for which platelets were stimulated with PGE1 in the presence and absence of ADP followed by fixation with formaldehyde. Following permeabilization with Triton X-100, VASP-P at serine 239 was determined using a monoclonal antibody (16C2) and a FITC-labelled secondary antibody, while platelets were counterstained with a PE-labelled anti-CD61 antibody. For dilutions, all antibodies of the test kit were used as by the manufacturer’s instructions. The PRI was calculated after measurement of VASP-P levels following stimulation with PGE1 (0.5 mol/L) by mean fluorescence intensity (MFI PGE1 ) as well as stimulation with PGE1 in the presence of ADP (20 mol/L, MFI PGE1 + ADP). The P2Y12 PRI is defined as: ([MFI PGE1 ] − [MFI PGE1 + ADP])/[MFI PGE1 ] × 100%, whereby background fluorescence is subtracted from each measurement. The lower the P2Y12 PRI is the better clopidogrel inhibits P2Y12 activity. For assessment of ranitidine interaction, the complete experiment was run in parallel in the presence of a pharmacologically relevant ranitidine concentration (400 ng/ml). 2.5. Substances Unless stated otherwise, chemicals were obtained from Sigma (Deisenhofen, Germany) in the highest purity available. 2.6. Statistics Individual measurements are shown. Statistical analysis was performed by one-way ANOVA followed by Tukey’s multiple comparisons test or a paired Student’s t-test where applicable; P < 0.05 was considered statistically significant. Data were analyzed using GraphPad Prism 4.0 (GraphPad Software, Inc., La Jolla, CA). 3. Results 3.1. Influence of H2 -receptor antagonism in platelets from healthy donors In PRP from healthy donors, ranitidine attenuated the aggregation-inhibiting effect of prostaglandin E1 (PGE1 ). While ADP-induced aggregation (91.6 ± 4.1%, trace 1 in Fig. 1A) was not significantly affected by ranitidine (92.5 ± 4.2%, trace 2 in Fig. 1A), PGE1 alone reduced ADP-induced aggregation (21.1 ± 1.8%, p < 0.001, trace 3 in Fig. 1A). Pre-incubation of PRP with the relevant concentration of ranitidine (400 ng/ml) led to an aggregation equivalent to that observed in the absence of both PGE1 and ranitidine (81.4 ± 9.0%, p < 0.001, trace 4 in Fig. 1A). Thus, blocking the adenylyl cyclase-stimulating H2 -receptor with ranitidine counteracts the platelet-inhibiting properties of PGE1 (Fig. 1A and B). 3.2. Influence of H2 -receptor antagonism in platelets from clopidogrel-treated patients Similar effects were observed in PRP obtained from clopidogreltreated CAD patients, in which pre-incubation with ranitidine abrogated the aggregation-inhibiting effect of PGE1 (Fig. 1C). While ADP-induced aggregation (41.2 ± 2.3%) was significantly reduced by PGE1 alone (29.5 ± 2.0%, p < 0.001), pre-incubation of PRP with the relevant concentration of ranitidine (400 ng/ml) led to an aggregation equivalent to that observed in the absence of both PGE1
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Fig. 1. Modulation of prostaglandin E1 (PGE1 )-mediated inhibition of ADP-induced maximal aggregation (Aggmax ) by ranitidine (Rani, 400 ng/ml) in PRP from clopidogrelnaïve healthy volunteers (A). Representative aggregation response curves from a healthy donor were recorded in the presence of ADP only (A, trace 1), ADP + RANI (A, trace 2), ADP + PGE1 (A, trace 3), and ADP + RANI + PGE1 (A, trace 4). The quantitative analysis is shown for healthy donors (n = 7, B) and for similar experiments in blood from clopidogrel-treated patients with coronary artery disease 24 h after receiving a 600 mg clopidogrel loading dose (n = 15, C).
Fig. 2. Adenosine-5 -diphosphate (ADP) activates platelets through two G protein-coupled P2 receptors, P2Y1 and P2Y12 . The G␣q -coupled P2Y1 receptor initiates ADPinduced aggregation via phospholipase C (PLC) and mobilization of calcium (Ca2+ ), while the G␣i -coupled P2Y12 receptor mediates amplification and completion of the aggregation response. In addition, P2Y12 is linked to the inhibition of cyclic adenosine monophosphate (cAMP) production by adenylyl cyclase (AC), which can be stimulated by prostacyclin (PGI2 ), prostaglandin E1 (PGE1 ) or histamine through specific Gs -coupled receptors, the isoprostane receptor (IP), PGE1 -receptor (EP2), and the histamine-2 receptor (H2 R), respectively. cAMP-activated proteinkinase A (PKA) phosphorylates the vasodilator-stimulated phosphoprotein (VASP), which inhibits platelet aggregation and secretion. The extent of ADP-induced attenuation of PGE1 -mediated VASP phosphorylation is used in the platelet-reactivity-index to determine P2Y12 -specific ADPactivity. Thienopyridines and new ADP-antagonists inhibit multiple pro-aggregatory actions of ADP, mostly by preventing the P2Y12 -mediated secondary ADP-response contributing to amplification of platelet activation. Blockade of H2 R suppresses stimulation of AC and leads to a shift of the AC pathway towards activation.
and ranitidine (41.5 ± 2.4%, p < 0.001). Similarly, addition of ranitidine enhanced ADP-induced platelet aggregation in blood from clopidogrel-treated patients (45.8 ± 2.3%, p < 0.01 vs. ADP in the absence of ranitidine). This indicates, that modulating H2 -receptor signaling, which is a Gs -protein-coupled receptor activating adenylyl cyclase and, thereby, counteracting P2Y12 (Fig. 2), can interact with clopidogrel efficacy on a platelet level. When adding pharmacologically relevant levels of ranitidine to blood samples from clopidogrel-loaded CAD patients, the PRI displaying specifically P2Y12 -dependent platelet reactivity was sig-
nificantly increased from 34.1 ± 5.5% to 44.1 ± 5.1% (p = 0.0005, Fig. 3). 4. Discussion In the present study we found that in platelets from patients on DAT, the H2 -receptor blocker ranitidine enhanced ADP-induced and P2Y12 -mediated platelet signaling. High variability in clopidogrel responsiveness has been widely described [17]. Using P2Y12 -specific assays to assess clopidogrel responsiveness such as the PRI, suboptimal responses in up to 60%
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Fig. 3. ADP-induced platelet signaling in clopidogrel-treated stable CAD patients (n = 15) 24 h after receiving a 600 mg loading dose in the absence and presence of a pharmacologically relevant concentration of ranitidine (400 ng/ml) was determined by ADP-mediated inhibition of PGE1 -induced VASP phosphorylation assessed by the PRI. The typical flow-cytometric histogram shows the maximum PGE1 -response (filled grey), the background signal (black curve), and the ADP-induced reactivity in the absence (blue curve) and presence (red curve) of ranitidine. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
of patients have been described [5]. In particular, a PRI >50% is associated with increased stent thrombosis and major adverse cardiovascular events [18–21]. Especially patients with high platelet reactivity despite receiving a 600 mg loading dose of clopidogrel had worse outcome after elective PCI [22]. Increasing clopidogrel dosage in patients undergoing PCI to achieve a PRI below 50% reduces subsequent cardiovascular events [23]. Since genetic loss-of-function of CYP2C19 is related to impaired pharmacokinetic and pharmacodynamic response to clopidogrel as well as impaired outcome [11–13], medications exerting profound CYP2C19 inhibition might reasonably be considered as a unfavorable option in patients on DAT. The PPI omeprazole is a profound inhibitor of CYP2C19 [24], and was identified in a prospective functional study to significantly interact with clopidogrel and reduce its platelet inhibitory effect [14]. In this double-blind placebo-controlled trial, patients undergoing PCI were randomized to receive either omeprazole or placebo for 7 days in addition to DAT. Omeprazole significantly decreased the ability of clopidogrel to inhibit platelet reactivity assessed by the P2Y12 -specific PRI [14]. Several retrospective observational studies showed conflicting results with regard to PPIs and clopidogrel. In a populationbased case–control study, current PPI use was associated with an increased risk of reinfarction, however, pantoprazole, which does not inhibit CYP2C19, had no association with readmission for myocardial infarction [25]. A retrospective cohort study of >8000 patients with ACS taking clopidogrel after discharge showed that concomitant use of clopidogrel and PPIs was associated with an increased risk of adverse outcomes than use of clopidogrel without PPIs. However, almost all patients using a single PPI throughout the observation period had been on omeprazole [4] Furthermore, one un-published analysis indicated a potential risk in all patients treated with any PPI [26], whereas more recent insurance data from other sources showed no such clear effect [27]. In a previous study, we observed no difference of clopidogrel responsiveness in stable CAD patients on clopidogrel maintenance therapy in the presence of pantoprazole [5]. Data from three retrospective evaluations (not even registries) prompted the FDA and EMEA to state their warnings with regard to combining PPIs with clopidogrel [4,25,26]. The retrospective analysis of an insurance database demonstrated the strongest influence
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of PPIs on outcomes, however, there was an enormous mismatch of patients taking PPIs vs. those not taking PPIs [26]. The PPI cohorts incorporated all known relevant clinical risk factors for impaired clopidogrel responsiveness [28]. Of interest, recent secondary analyses from large randomized controlled trials comparing clopidogrel vs. active competitors in more homogenous patient collectives did not find differences in clinical outcomes when comparing PPItreated vs. not PPI-treated patients [29]. From an underpowered group in one of the insurance databases, it was actually recommended to substitute PPIs with H2 -receptor blockers for gastric ulcer prevention [26]. Again FDA and EMEA both stated that there were no data indicating an interaction. However, literature research revealed a paper describing that concomitant ranitidine treatment in acetylsalicylic acid-treated patients enhances ADP-induced aggregation, which is intended to be blocked by clopidogrel, and reduces circulating salicylate levels [30], thereby potentially interacting with both components of DAT. Indeed, our in vitro data on P2Y12 -specific signaling demonstrates that ranitidine interacts with P2Y12 -mediated signaling on the platelet level (Fig. 1), thereby contributing to impaired functional clopidogrel-responsiveness (Fig. 3). One P2Y12 -specific signaling of ADP is the inhibition of adenynyl cyclase through a G␣i . Adenynyl cyclase is constitutively stimulated by endogenous platelet inhibitors such as prostacyclin and prostaglandin E1 through Gs -protein-coupled receptors. Histamine acts on platelets through a similar Gs -protein-coupled receptor, the H2 receptor. By blocking this receptor, the balance is shifted towards less stimulation of adenylyl cyclase and, therefore, an ADP-like effect (Fig. 2). Due to the mechanism of P2Y12 -mediated and H2 -mediated signaling, a clopidogrel-counteracting effect of ranitidine is not surprising. This mechanism is absolutely distinct from potential effects of ranitidine by heightening gastric pH leading to reduced release of clopidogrel from the oral formulation or potential interactions with hepatic clopidogrel metabolism, which were beyond the scope of this investigation. Furthermore, a recent pharmacological assessment described that ranitidine affected the pharmacokinetics of clopidogrel maintenance dosing as well as prasugrel loading in patients treated with ranitidine [31]. It can be assumed, that other H2 -receptor blockers will have the same effect on the platelet level, however, cimetidine should not be considered as an appropriate substitution for PPIs, as its CYP2C19 inhibiting properties were even mentioned in the EMEA warnings. In conclusion, we demonstrate that ranitidine, which has been suggested as an alternative to PPIs in patients on DAT, does interfere with clopidogrel on the platelet level and should, therefore, not be recommended in the absence of prospective data as a comedication to clopidogrel.
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